Intraluminal chloride regulates lung branching morphogenesis: involvement of PIEZO1/PIEZO2.

BACKGROUND: Clinical and experimental evidence shows lung fluid volume as a modulator of fetal lung growth with important value in treating fetal lung hypoplasia. Thus, understanding the mechanisms underlying these morphological dynamics has been the topic of multiple investigations with, however, limited results, partially due to the difficulty of capturing or recapitulating these movements in the lab. In this sense, this study aims to establish an ex vivo model allowing the study of lung fluid function in branching morphogenesis and identify the subsequent molecular/ cellular mechanisms. METHODS: Ex vivo lung explant culture was selected as a model to study branching morphogenesis, and intraluminal injections were performed to change the composition of lung fluid. Distinct chloride (Cl-) concentrations (5.8, 29, 143, and 715 mM) or Cl- channels inhibitors [antracene-9-carboxylic acid (A9C), cystic fibrosis transmembrane conductance regulator inhibitor172 (CFTRinh), and calcium-dependent Cl- channel inhibitorA01 (CaCCinh)] were injected into lung lumen at two timepoints, day0 (D0) and D2. At D4, morphological and molecular analyses were performed in terms of branching morphogenesis, spatial distribution (immunofluorescence), and protein quantification (western blot) of mechanoreceptors (PIEZO1 and PIEZO2), neuroendocrine (bombesin, ghrelin, and PGP9.5) and smooth muscle [alpha-smooth muscle actin (α-SMA) and myosin light chain 2 (MLC2)] markers. RESULTS: For the first time, we described effective intraluminal injections at D0 and D2 and demonstrated intraluminal movements at D4 in ex vivo lung explant cultures. Through immunofluorescence assay in in vivo and ex vivo branching morphogenesis, we show that PGP9.5 colocalizes with PIEZO1 and PIEZO2 receptors. Fetal lung growth is increased at higher [Cl-], 715 mM Cl-, through the overexpression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and α-SMA. In contrast, intraluminal injection of CFTRinh or CaCCinh decreases fetal lung growth and the expression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and α-SMA. Finally, the inhibition of PIEZO1/PIEZO2 by GsMTx4 decreases branching morphogenesis and ghrelin, bombesin, MLC2, and α-SMA expression in an intraluminal injection-independent manner. CONCLUSIONS: Our results identify PIEZO1/PIEZO2 expressed in neuroendocrine cells as a regulator of fetal lung growth induced by lung fluid.

ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: Characterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2+) and distal (SOX9+) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance. METHODS: The expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and ß-Catenin were quantified by Western blot. RESULTS: Experimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho ß-Catenin, and BMP4. CONCLUSIONS: These studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

Distinct Epithelial Cell Profiles in Normal Versus Induced-Congenital Diaphragmatic Hernia Fetal Lungs.

Background: Recent studies identified a great diversity of cell types in precise number and position to create the architectural features of the lung that ventilation and respiration at birth depend on. With damaged respiratory function at birth, congenital diaphragmatic hernia (CDH) is one of the more severe causes of fetal lung hypoplasia with unspecified cellular dynamics. Objectives:  To characterize the epithelial cell tissue in hypoplastic lungs, a careful analysis regarding pulmonary morphology and epithelial cell profile was conducted from pseudoglandular-to-saccular phases in normal versus nitrofen-induced CDH rat lungs. Design: Our analysis comprises three experimental groups, control, nitrofen (NF) and CDH, in which the relative expression levels (western blot) by group and developmental stage were analyzed in whole lung. Spatiotemporal distribution (immunohistochemistry) was revealed by pulmonary structure during normal and hypoplastic fetal lung development. Surfactant protein-C (SP-C), calcitonin gene-related peptide (CGRP), clara cell secretory protein (CCSP), and forkhead box J1 (FOXJ1) were the used molecular markers for alveolar epithelial cell type 2 (AEC2), pulmonary neuroendocrine, clara, and ciliated cell profiles, respectively. Results: Generally, we identified an aberrant expression of SP-C, CGRP, CCSP, and FOXJ1 in nitrofen-exposed lungs. For instance, the overexpression of FOXJ1 and CGRP in primordia of bronchiole defined the pseudoglandular stage in CDH lungs, whereas the increased expression of CGRP in bronchi; FOXJ1 and CGRP in terminal bronchiole; and SP-C in BADJ classified the canalicular and saccular stages in hypoplastic lungs. We also described higher expression levels in NF than CDH or control groups for both FOXJ1 in bronchi, terminal bronchiole and BADJ at canalicular stage, and SP-C in bronchi and terminal bronchiole at canalicular and saccular stages. Finally, we report an unexpected expression of FOXJ1 in BADJ at canalicular and saccular stages, whereas the multi cilia observed in bronchi were notably absent at embryonic day 21.5 in induced-CDH lungs. Conclusion: The recognized alterations in the epithelial cell profile contribute to a better understanding of neonatal respiratory insufficiency in induced-CDH lungs and indicate a problem in the epithelial cell differentiation in hypoplastic lungs.

Imagiological methods for prediction of fetal pulmonary hypoplasia: a systematic review.

OBJECTIVE: To compare the different imagiological methods for prediction of fetal pulmonary hypoplasia (PH) in general diseases and associated with preterm rupture of membranes (PROM), and congenital diaphragmatic hernia (CDH). METHODS: According to PRISMA guidelines, all the literature on PH from 1988 to 2018 was reviewed. Twenty-nine articles were selected and analyzed for two- and three- dimensional-ultrasounds (2D and 3D-US) as predictors for the lethal outcome. RESULTS: Overall, the results identify a general gap correlating prenatal pulmonary measurements and survival at birth; discrepant predictive values for the same imagiological methods are explained by the group heterogeneity in terms of diseases and degree of severity, with the 2D measurements being more affected than 3D; 2D and 3D-US present equally predictive values for groups with 0% of survival. Regarding PROM, results demonstrate comparable accuracies for similar survival rates suggesting a useful predictive value of 2D-US in outcome estimation; they also identify ultrasonographic methods as a more accurate prognostic factor than gestational age at rupture, latency or amniotic fluid index. In CDH, consistent with previous studies, our review shows magnetic resonance imaging as a better survival predictor followed by the 3D and 2D methods, while 2D-LHR was the more precise prognosticator correlating prenatal PH, survival at birth, and the need for neonatal respiratory support. CONCLUSION: Ultrasonographic methods can be valuable predictors for lethal PH and should be validated for a broad set of diseases (e.g. PROM). For that, restricted studies for disease groups and correlating fetal PH with the needed of neonatal support, and survival at birth is critically recommended.

MicroRNA processing machinery in the developing chick embryo.

Gene expression regulation during embryo development is under strict regulation to ensure proper gene expression in both time and space. The involvement of microRNAs (miRNA) in early vertebrate development is documented and inactivation of different proteins involved in miRNA synthesis results in severe malformations or even arrests vertebrate embryo development. However, there is very limited information on when and in what tissues the genes encoding these proteins are expressed. Herein, we report a detailed characterization of the expression patterns of DROSHA, DGCR8, XPO5 and DICER1 in the developing chick embryo, from HH1 (when the egg is laid) to HH25 (5-days incubation), using whole mount in situ hybridization and cross-section analysis. We found that these genes are co-expressed in multiple tissues, mostly after stage HH4. Before early gastrulation DICER1 expression was never detected, suggesting the operation of a Dicer-independent pathway for miRNA synthesis. Our results support an important role for miRNAs in vertebrate embryo development and provide the necessary framework to unveil additional roles for these RNA processing proteins in development.